1. Field of the Invention
This invention relates to therapeutic agents for use in the treatment of mammalian, particularly human, autoimmune diseases. The invention also relates to therapeutic agents useful in the treatment of human leukaemias of a T cell origin, as so-called “vaccine carriers”, and as agents for use in the prevention of human transplantation rejection and graft versus host disease (GVHD).
2. Description of the Related Art
In an article entitled “Morphologic and Functional Alterations of Mucosal T Cells by Cholera Toxin and its B subunit” by Charles O. Elson et al., The Journal of Immunuology, 1995, 154; 1032-1040 it is disclosed that the cholera toxin (Ctx) and the CtxB subunit inhibit CD8+ and CD4+ T cells.
Reference is also made to the paper entitled “Prevention of Acute Graft-Versus-Host Disease by Treatment with a Novel Immunosuppressant” by B. Yankelevich et al., The Journal of Immunology, 1995, 154: 3611-3617. This identifies CtxB as an agent for use in bone marrow transplantation for the prevention of acute graft-versus-host disease (GVHD). WO 95/10301 discloses an immunological tolerance-inducing agent comprising a mucosa-binding molecule linked to a specific tolerogen.
As used herein, the term “Ctx” refers to the cholera toxin and “CtxB” to the B subunit of the cholera toxin. In other texts, these may sometimes be identified as “CT” or “Ct” and “CTB” or “CtB” respectively. The term “Etx” herein means the E. coli heat labile enterotoxin, and “EtxB” is the B subunit of Etx. In other texts, these may sometimes be identified as “LT” or “Lt” and “LtB” or “LtB” respectively.
E. coli enterotoxin. As mentioned above, the term. Etx refers to the heat labile E. coli enterotoxin which is a member of a family of toxins that includes cholera toxin and which cause diarrhoeal diseases in humans and domestic animals. Such toxins are comprised of two components, the so called A and B-subunits. The ability to cause diarrhoea resides with properties of the A-subunit, which is an enzyme capable of increasing the concentration of the biochemical second messenger cyclic AMP within epithelial cells lining the intestine. The rise in cyclic AMP levels leads to the loss of ions from the cells and the consequent loss of water which causes diarrhoea. The B-subunit moiety has evolved in order to deliver the A-subunit into epithelial cells by a process which involves it binding to a membrane located glycolipid receptor, GM1. The B-subunit is itself not toxic, and is unable to cause diarrhoea.
EtxB. The B-subunit is composed of five individual polypeptides bound tightly together into a doughnut ring like structure. Each polypeptide contains a site for interaction with GM1, and thus exposure of cells to EtxB causes cross-linking of GM1 at the cell surface. The overall size of EtxB is 60 kilodaltons, with each of the five polypeptides being composed of 103 amino acids. Its exceptional stability results from the very close association of interfaces between adjacent polypeptides which form the B-subunit pentamer. Thus EtxB is stable as a pentamer under conditions which would lead to disruption of normal protein structure. This stability is reflected by the observation that the pentamer remains intact at 84° C., between pH 2 and pH 11, and in the presence of ionic detergents and proteolytic enzymes. This makes EtxB one of the most stable protein complexes known and may facilitate ease of use as a component in human medicines.
The key finding that EtxB can alter immune responses has come from investigations of its binding to GM1 on cells other than those of the intestinal epithelium. GM1 is found on all cells of the immune system, and its cross-inking by EtxB triggers signals which can alter their activity. Administration of EtxB either into the nose, by mouth, or by injection, can alter the local environment within which immune responses are triggered. This facilitates the production of high levels of antibodies against antigens of infectious agents which are mixed with EtxB, and can cause the down-regulation of the damaging inflammatory responses which are associated with autoimmunity. Thus, EtxB may be used: alone in the treatment of autoimmune disease (as an immunotherapeutic), or in combination with vaccine antigens (where it acts as an adjuvant). The ability to act as an adjuvant following mucosal delivery makes the B-subunit almost unique since most infectious agents gain access to the body through these surfaces, and injected vaccines do not stimulate strong responses at such sites.
The immunological mechanisms underlying the use of the B-subunit. The B-subunits ability to modulate the immune response is dependent on its capacity to modulate the activity of T-cells, B-cells and populations of antigen presenting cells. Each of these cell types plays a critical role in the development of the immune response. In the normal response to a foreign organism, antigens are internalised by antigen presenting cells, of which dendritic cells are the most important. These cells are specialised in breaking down proteins into short amino acid sequences (peptides) which associate with major histocompatibility complex (MHC) molecules which are then transported to the cell surface.
Foreign peptides bound to class II MHC molecules are recognised by T-helper cells (CD4+ T-cells) which are activated as a result and begin to divide, differentiate and secrete hormone-like messengers called cytokines. The T-helper cells then co-ordinate and maintain the immune response. Subsequent responses can involve the activation of i) B-cells which mature into plasma cells capable of producing antibodies, ii) macrophages and neutrophils which enter the sites of infection and ingest foreign material leading to its destruction, and iii) other types of T-cell (CD8+ T-cells) which can recognise virally infected cells of the body and kill them.
Most normal immune responses will involve activation of all of these components to some extent, however, it is clear that certain factors can affect which particular components are dominant. Further, in certain circumstances it is clearly beneficial to be able to tailor which type of response is elicited. For example, in preventing infection at mucosal surfaces, it is desirable to stimulate a strong antibody response, but avoid the activation of macrophages and neutrophils which can themselves cause inflammation and tissue damage.
In order to co-ordinate different types of immune response, humans have evolved the capacity to sense the nature of the foreign challenge and alter the T-helper cell response accordingly. Thus, T-helper cells can be distinguished as being either T-helper 1 (Th1) or T-helper 2 (Th2) cells. Th1 cells secrete cytokines including gamma interferon (IFNγ) and interleukin (IL)-2 which activate macrophages, neutrophils and CD8+ T-cells and which lead to the production of antibodies which promote inflammation. In contrast, Th2 cells secrete IL-4, IL-5, IL-6 and IL-10, down-regulate Th1 responses and promote the production of antibodies which are secreted at mucosal surfaces, or which do not trigger inflammation. In addition to the cross-regulation of Th1 responses by Th2 cells and vice versa, it is also clear that other CD4+ T-cell populations are induced which down-regulate both types of response (T-regulatory cells). In, for example, an immune response to a virus which infects the eye, it is desirable to trigger a strong Th2 response which can arm the local tissue with antibodies to block the infection, while avoiding stimulating Th1 responses which will themselves cause damage to such a delicate tissue.
Autoimmune disease results when the bodies own processes of regulation breakdown. In these cases components of the body are mistakingly identified as ‘foreign’ and an immune response is mounted which attacks the individuals own tissues. In the majority of examples of autoimmune disease, tie immune response is driven by Th1 cells which cause macrophages and neutrophils to enter and disrupt the tissue. For example, in the case of rheumatoid arthritis an immune response to joint-associated antigens leads to the chronic infiltration of neutrophils and macrophages which cause cartilage and bone degradation, pain, swelling and loss of function. Further, type 1 diabetes results from a similar process leading to the destruction of insulin producing islets within the pancreas. The precise reasons for the loss of regulation within the immune systems of certain individuals are not clear, but certainly involve complex genetic and environmental factors.
The B-subunit influences the processes involved in antigen recognition by T-helper cells. In doing so it can promote the activation of Th2 and T-regulatory cells, while at the same time suppressing the activation of Th1 cells. Consequently, EtxB may be used to treat Th1-driven autoimmune diseases, or may be added to antigens derived from infectious agents to trigger production of large quantities of protective mucosal and serum Th2-associated antibodies. Importantly, EtxB does not promote the production of IgE antibodies which are the cause of allergy.
The B-subunit alters the balance between Th1 and Th2 immune responses. It is clear that the local microenvironment in which antigen is presented to CD4+ T-cells determines the nature of the subsequent response (FIG. 12). Certain factors can promote the differentiation of T-helper cells into Th1 cells, while others cause differentiation into Th2 cells or T-regulatory cells. Evidence indicates that EtxB influences many of these events. The differentiation of T-helper cells into Th1 cells is promoted by cytokines produced by antigen presenting cells themselves (in particular, dendritic cell and macrophage derived IL-12) as well as CD8+ T-cells (producing gamma interferon).
The present inventors have established that EtxB inhibits the production of IL-12 by antigen presenting cells (FIG. 13) and removes CD8+ T-cells by causing them to die by apoptosis (FIG. 14). Therefore, EtxB interferes with the two major factors which promote the development of pro-inflammatory Th1 responses. The differentiation of T-helper cells into Th2 cells is promoted by their interaction with B-cells during the activation process, and by the secretion of IL-10 from antigen presenting cells. IL-10 is also thought to play a critical role in the generation and activities of T-regulatory cells. The present inventors have shown that EtxB activates B-cells leading to their enhanced interaction with T-helper cells (FIG. 15), and causes the production of IL-10 by antigen presenting cells (FIG. 16). Thus, EtxB enhances the presence of the two major factors which promote the activation of Th2 cells and creates conditions which favour the induction of regulatory cell populations.
Taken together, the activities of EtxB allow it to shift the balance of the immune response suppressing the Th1 arm while promoting the Th2 and T-regulatory anus (FIG. 12). Thus, EtxB can turn off the damaging inflammation in autoimmunity, and can trigger the production of non-inflammatory antibodies in the serum and at mucosal surfaces.
The B-subunit can be used to prevent or treat autoimmune disease. The ability of EtxB to intervene in the processes underlying autoimmune disease has been tested in animal models of arthritis. Usefulness in the treatment or prevention of arthritis has been established using a widely recognised mouse model in which disease is induced in male DBA/1 mice by the injection of type II collagen in complete Freunds adjuvant (collagen-induced arthritis). Mixing a joint antigen in this way with an adjuvant which triggers strong Th1 responses leads to a strong pro-inflammatory immune response against collagen. This response is characterised by the appearance of anti-collagen antibodies and demonstrable T-cell reactions. By day 20 after induction, joint swelling becomes apparent and its incidence and severity continues to increase until approximately day 45 at which point 70-80% of animals have some swelling, usually involving the hind ankle and knee joints. Damage is also occasionally noted in the forelimbs. Like rheumatoid arthritis in humans, the model is characterised by joint swelling and histological signs of macrophage and neutrophil infiltration into the joint space. In addition, cartilage and bone destruction are a common feature along with the formation of a pannus. Assessment of the immune response to collagen in mice with arthritis reveals the presence of pro-inflammatory antibodies as well as high levels of the Th1 cytokine, gamma interferon, in cultures of lymph node T-cells.
Collagen induced arthritis (CIA) can be prevented by treatment with EtxB alone. When EtxB is given to mice intranasally, orally or by injection prior to the collagen injection, it can block the induction of disease as revealed by a reduction in clinical joint swelling (FIG. 17) and histological damage (FIG. 18). In the experiments shown, EtxB was administered into the nose or by mouth on four occasions daily up to the day of collagen injection. Injected EtxB was given on a single occasion on the day of collagen challenge.
In each case the dose of EtxB used was 100 μg. Investigations have shown that in the case of nasal delivery identical levels of disease protection can be achieved with 10 μg, or 1 μg. The delivery of EtxB led to a dramatic reduction in pro-inflammatory anti-collagen antibody levels and led to a shift in the nature of the T-helper cell response away from the production of gamma interferon.
The B-Subunit can be used as a potent mucosal vaccine adjuvant. The need to potentiate immune responses to vaccine antigens is widely recognised. At present the only adjuvant licensed for human use is alum, which is given by injection and fails to elicit significant mucosal antibody production. Given that the majority of infectious disease causing organisms enter across mucosal surfaces, it is clear that an effective mucosal adjuvant will have widespread applications. The present inventors have shown that EtxB is a highly potent adjuvant which can stimulate strong immunity to foreign antigens after mucosal administration. Important work has been carried out by the present inventors (see WO 99/58145) using a mouse model of herpes simplex virus type 1 (HSV-1) infection of the eye.
Herpes simplex virus type 1 infection usually occurs in early childhood and affects over 90% of adults. The initial infection is usually unnoticed, although occasionally can cause severe disease. However, following initial infection the virus resides, dormant, in the nervous system which supplies the face and eyes. In some people HSV can reactivate and leads to disease in any of these areas. On the skin HSV-1 causes ‘cold sores’ which are usually self-limiting, although unpleasant. However, when virus enters the eye, it causes ulcers on the surface of the eye which can result in blindness and the need for corneal transplantation. HSV-1 is the major cause of post-infection blindness in the developed world. A close relative of HSV-1, HSV-2, is associated with similar, although more severe, infections of the genital tract.
When a glycoprotein mixture from HSV-1 is given to alone to mice intranasally, it fails to induce an immune response to HSV. By contrast, the addition of EtxB to this glycoprotein mixture potentiates a very strong response involving the secretion of large amounts of anti-HSV antibody into the serum and at mucosal surfaces (FIG. 21). The antibody response is clearly evident both at the eye as well as at distant sites including the vagina. The underlying T-helper cell response to HSV is associated with the production of predominantly Th2 cytokines. The immune response induced using EtxB as a mucosal adjuvant can protect mice against a severe ocular HSV-1 infection (FIG. 22a). In the model, the vaccine, when given prior to challenge with HSV-1 reduces the severity of symptoms in the eye and prevents the high levels of viral spread to the central nervous system (a rare complication in humans). These findings demonstrate that EtxB can be used as an adjuvant for a prophylactic vaccine against HSV-1.
In further studies, the present inventors have shown that EtxB can be used to develop a therapeutic vaccine against HSV-1. In these experiments mice were infected with HSV-1 and left in order to allow the virus to become dormant in the nervous system prior to vaccination. Intranasal administration of HSV-1 glycoproteins with EtxB altered the nature of the existing response to HSV-1 in these animals such that they showed markedly reduced levels of disease following reactivation of the virus using a physiological stimulus to the eye (FIG. 22b). This additional finding highlights the potential of EtxB as a critical component allowing therapeutic vaccination.
These findings demonstrate that intranasal administration of a vaccine containing EtxB as an adjuvant induces immunity at local and distant mucosal sites. A similar intranasal vaccine using glycoproteins from HSV-2 may be effective in stimulating protective immunity against genital infection. Further, the profile of the immune response stimulated using EtxB is of clear relevance to protection against a wide range of infectious agents. Indeed, experiments have shown that EtxB may be used to stimulate immunity against a broad range of other antigens.
The B-subunit can be used to target the delivery of peptides into cells. The effective induction of cytotoxic T-cell responses requires the entry of antigens into the cytosol of antigen presenting cells. While some externally added soluble antigen may enter this compartment, targeted delivery into the cytosol should augment the induction of this component of the immune response. Cytotoxic T-cell responses are particularly important in facilitating the removal of infectious agents which reside within cells of the body, such as viruses and certain bacteria. Thus, in some vaccines the ability to augment the cytotoxic T-cell response as well as stimulate high levels of antibodies would be beneficial. To achieve this, an efficient delivery system which results in the targeting of antigens into the cytosol is required. The B-subunit exhibits characteristics which indicate that it may function in this way.
Cross-linking of GM1 by the B-subunit is followed by its internalisation into vessicles within the cell. This capacity to enter cells has been used to deliver attached peptides into the cytosol. The present inventors have demonstrated that peptides ranging from nine to twenty seven amino acids in length can be genetically or chemically conjugated to the B-subunit without interfering with its stability or ability to bind to GM1. Addition of such conjugates to cells results in the liberation of the attached peptides within a vessicular compartment and their subsequent delivery to the cytosol. Delivery of EtxB in this way may lead to the presentation of the peptides to stimulate the activation of cytotoxic T-cells.
The B-Subunit is a Lead Compound to the Development of Small Molecule Mimetics.
All of the described effects of EtxB are dependent on its ability to bind to the cell surface. The present inventors have demonstrated in WO 00/14114 that a mutant B-subunit that is unable to bind to the cell surface is completely ineffective. A refinement of this approach has identified a loop with the B-subunit which is responsible for triggering the effects on immune cells. Surprisingly, this loop is not directly involved in allowing binding to GM1, indicating a critical role for interaction with a secondary receptor at the cell surface. A synthetic peptide corresponding to this loop exhibits a similar capacity to modulate T-cell function in vitro. This observation indicates that the loop represents a lead compound for small molecule mimetic chemistry which may allow the development of higher affinity analogues for use as immunotherapeutics and vaccine adjuvants.
Diabetes. Insulin dependent diabetes mellitus (IDDM) is an autoimmune disease resulting form the T-cell dependent destruction of insulin-producing cells from the pancreas Langerhans islets (1). It affects about 4 million people in Europe and North America alone and usually appears before the age of 30. There is no cure. Sufferers must give themselves daily insulin injections to control their blood glucose levels. It is unclear what triggers the immune system's attack on the islet cells because the regulation of the auto-aggressive immune response is complex, resulting from the interaction between several T cell subsets and their activation of mononuclear phagocytes. Islet destruction, both in humans and rodents, is attributed to the existence of auto-reactive CD4+T cells that recognise islet antigens and belong to the Th1 subset (i.e. secrete inflammatory cytokines such as IFNγ) (2). Such cells could be isolated from diabetic rodent spleens or pancreas inflammatory infiltrates and transferred the disease to syngenic receipents (3).
The present invention seeks to provide an improved mechanism for preventing and treating IDDM.